All organisms strive to maintain genomic fidelity in the face of agents that can damage their genetic material and the possibility that errors that can occur whenever their DNA is replicated. The ultimate goals of my research are to understand (i) how the mechanism and high-level coordination of DNA repair processes are governed by molecular, genetic, and epigenetic factors in vivo; (ii) how these factors affect diverse repair processes in different contexts to affect human health; and (iii) how clinically-important modulators of DNA repair activities and of repair-related toxicity can be leveraged as novel therapeutics. I have focused primarily on DNA mismatch repair (MMR) pathways, the pathways responsible for correcting errors that occur during DNA replication. As a primary mechanism of mutation avoidance in nearly all organisms, MMR plays a central role in many diverse processes that affect human health, from the emergence of drug resistance in infectious pathogens and cancers to the onset and treatment of somatic genetic diseases. We developed a novel assay to deconstruct the biomolecular mechanisms of MMR that uses chemically-modified oligonucleotide probes to insert targeted DNA `mismatches' directly into the genome of living cells. This assay, which we call by the acronym `SPORE,' can thus be used to directly interrogate replication-coupled repair processes like MMR quantitatively in a strand-, orientation-, and lesion-specific manner in vivo?something nearly impossible to achieve otherwise. Using the SPORE assay as a uniquely powerful baseline of approach, and in combination with next-generation biotechnologies like CRISPR and innovative experimental design, my laboratory will seek to answer the following broad-spectrum and transdisciplinary questions: How do different molecular, genetic, and epigenetic factors affect the higher-order architecture (components and interactions), coordination, dynamics of different MMR mechanisms? How do these factors affect repair-associated toxicities? Are different molecular lesions recognized by MMR repaired according to different mechanisms and toxicities? Do the unique repair mechanisms in pathogenic organisms represent a novel source of antimicrobial targets? How do viral factors and environmental mutagens modulate MMR and MMR-related toxicities and by what mechanism? What is their role in hypermutation and emergence of drug resistance? What governs the tradeoff between mutagenic and anti-mutagenic roles of MMR in microsatellite instability (MSI) diseases? What occurs during collisions between DNA repair or other processes on DNA, and what is the nature and origin of related catastrophic mutational events? These questions are each complex in their own right and have remained difficult to answer using traditional techniques, but our unique hybrid approach provides a direct way to address each of them. The likely outcomes during the R35 award will be numerous breakthroughs in our understanding of mutational processes and how it can be manipulated in living cells; with a long-term impact being a sea-change in the ability to probe and exploit DNA damage repair mechanisms to treat disease.